Chalkiness and premature controlled by energy homeostasis in OsNAC02 Ko-mutant during vegetative endosperm development

Background Chalkiness is a common phenotype induced by various reasons, such as abiotic stress or the imbalance of starch synthesis and metabolism during the development period. However, the reason mainly for one gene losing its function such as NAC (TFs has a large family in rice) which may cause premature is rarely known to us. Results The Ko-Osnac02 mutant demonstrated an obviously early maturation stage compared to the wild type (WT) with 15 days earlier. The result showed that the mature endosperm of Ko-Osnac02 mutant exhibited chalkiness, characterized by white-core and white-belly in mature endosperm. As grain filling rate is a crucial factor in determining the yield and quality of rice (Oryza sativa, ssp. japonica), it's significant that mutant has a lower amylose content (AC) and higher soluble sugar content in the mature endosperm. Interestingly among the top DEGs in the RNA sequencing of N2 (3DAP) and WT seeds revealed that the OsBAM2 (LOC_Os10g32810) expressed significantly high in N2 mutant, which involved in Maltose up-regulated by the starch degradation. As Prediction of Protein interaction showed in the chalky endosperm formation in N2 seeds (3 DAP), seven genes were expressed at a lower-level which should be verified by a heatmap diagrams based on DEGs of N2 versus WT. The Tubulin genes controlling cell cycle are downregulated together with the MCM family genes MCM4 ( ↓), MCM7 ( ↑), which may cause white-core in the early endosperm development. In conclusion, the developing period drastically decreased in the Ko-Osnac02 mutants, which might cause the chalkiness in seeds during the early endosperm development. Conclusions The gene OsNAC02 which controls a great genetic co-network for cell cycle regulation in early development, and KO-Osnac02 mutant shows prematurity and white-core in endosperm. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04845-8.

(b-1) Ko-Osnac02 mut construction with two mutant sites in OsNAC02 CDS.The genetic type of mutants are labeled in red and the PAM "GGA" is highlighted in bold and underline.The mutant with "ATTGCTC" 7 bp-deletion in N2 mut located at 399-421 bp of OsNAC02 sequence, and the mutant with "A→C" at 1350 bp, " A→T " / " A→x " transversion at 1360 bp of OsNAC06 sequence.(b-2) N3 mut constructed the mutant sites in OsNAC02 / OsNAC06 CDS with PAM "GGA"/ "TGG" respectively.The Crispr-Cas9 induced "T" deletion in N2 and "T" insertion and 3 bp -"GGC" deletion in N3 at 1256-1278bp region respectively.(c) As these figures shows, the mutant sites of N2/N3 mut in OsNAC02 CDS detected by the alignment with DNA sequence of WT (NIP).(f) The weight per panicles (95DAG) of N2/N3 mutant are detected, and there is no significant difference between mutants and WT.Five panicles from each plant are selected randomly for detection respectively.(g) The number of seeds per panicle in T1 generation (95DAGs) was detected, and N2 mut has most seeds per plant.This phenotype together was detected with the number of panicles together in the same materials.
All the mutants planted in field under the normal conditions (Fuyang, Hangzhou in 2021),the statistic analysis are running by the student t-test,*, p < 0.05 and **, p < 0.01.The T0 generation of transplants was planted in Lingshui (Hainan Province Island ,18.5°110'E) in the winter of 2020, and we obtained the T1 generation.Following the T1 generation was planted in Fuyang (Hangzhou in Zhejiang Province, 30°120'E) during the summer in 2021, and T2 generation were harvested in the autumn of 2022.The OsNAC02 (Os02g0594800，LOC_Os02g38130) and OsNAC06 (Os06g0267500) are labeled by bule circle.

As the yield characters shown in
The two genes are belonged to subgroup III and IV respectively.
The phylogenetic tree of OsNAC family was constructed using the MEGA 7.0.and the NAM regions in all genes are highlighted by TBtools11.2.The green boxes in the phylogenetic tree are NAC domains of genes.

Fig.S3
The homologous clusters of OsNAC family genes by phylogenetic analysis The homologous analysis of NAC in plant (a) The homologous analysis of NAC proteins with amino acid sequence alignment in the rice, maize and Arabidopsis by using DNAMAN 7.0.(b) The Uniprot accession of 9 genes in homologous analysis.
(https://www.uniprot.org/).OsNAC06 (Os06g0267500) has simple protein constructure and contains only 246 aa, which was highly homologous to AtSOG1.Its N-terminal region with 0 -45aa was a strictly conserved region of NAM domain.

Fig. S1
Fig.S1 The mutant tags of target sites and mutants detection by PCR sequencing b-2 Fig.S2The yield characters of N2 / N3 mut in field(95DAG in 2021, 125DAG in 2020)

Fig 1 ,
the shape phenotype in seeds (125 DAG) of N2/N3 mutants are no difference compared with the control (NIP).
Fig.S6 The chalky phenotype of N2/N3 mut seeds (T2) in 2021 (a)The chalky phenotypes of the milled rice are calculated by chalky ratio and the chalky area .The transparent are low in both mutant and WT, but the chalky ratio in mutants are obvious higher than that in WT.The rice broken ratio of N3 mut is highest of all.All the statistic analysis were performed by running a two-way ANOVA on the mixed model, *, p < 0.05, **, p < 0.01 and ***, p < 0.001.(b) The sample names are WT, N2 and N3 in the figure respectively.As the chalky phenotypes of milled rice and broken ratio of N2 and N3 mut per plant shown in this figure, both N2 and N3 are core-white in milled rice, but only N2 has core and belly white in its floury endosperm.As these figures show the yield per plant simultaneously, only N3 has highest yield and broken ratio in the milled rice per plant.n=10 mm.(c) The three phenotypes of transparent in N2 and N3 mutant by statistics analysis.
Fig.S8 PCA clusters based on RNA-seq analysis The trend of PCA analysis are based on the RNA-seq analysis of seed (3 DAP),and four samples are clustered into 3 PCA groups:The red spot was PCA1 cluster with WT and N2-1 (N2-x); The yellow spot was PCA2 cluster with N2-2 (N2-y) ,which used as duplicate sample of N2-x for the KO-Osnac02 mut ; The blue spot was PCA3 cluster with N3_1 only.
The ABRE/ G-box motif locations on the promoter regions of OsNAC02 regulated genes Table the motif analysis of genes regulated by OsNAC02 in Co-network

Table
Chalky phenotype of mutants by the statistics analysis (%)